Previous in vivo electrophysiological studies across differing brain regions demonstrated ethanol enhancement of GABA responses from some, but not all, neurons. This differential effect of ethanol was attributed to a variable action on diverse GABAA receptor subtypes. In contrast to ethanol's consistent enhancement of GABA function in vivo, GABA responses from isolated neurons in vitro were rarely enhanced by ethanol (Preliminary Data). Based upon the contrast of our in vivo and in vitro results, it was proposed that a factor, not available to isolated neurons in vitro, allowed ethanol's action in vivo. Based upon the presence of auxiliary binding sites on GABAA receptors, it was hypothesized that endogenous compounds acting on one or more of the auxiliary sites were important for ethanol to alter the action of GABA responsiveness in vitro. In support of this explanation, our laboratory recently discovered that ethanol enhanced GABA responses from most dissociated substantia nigra reticulata (SNR) neurons in the presence of a neurosteroid agonist, alphaxalone, while having no effect on GABA responses in the absence of this neurosteroid (Preliminary Data). Therefore, the present proposal will test the hypothesis that activation of specific neurosteroid sites on GABAA receptors facilitates ethanol enhancement of GABA responses from isolated neurons. Four Specific Aims will test this view. In Specific Aim I, several classes of neurosteroids will be examined for their ability to allow ethanol enhancement of GABA activity from isolated SNR cells. It is assumed that the differing neurosteroids will not have identical effects on the action of ethanol to enhance GABA responses. Specific Aim II will determine if a GABA-enhancing neurosteroid will result in ethanol having a differential influence on responsiveness to GABA from neurons isolated from selected brain regions where ethanol in vivo has varying effects on GABA function. Specific Aim III will determine the mRNAs for the GABAA receptor subunits in individual neurons sensitive and insensitive to ethanol's action to enhance GABA responses in the presence of neurosteroid. From the mRNAs in individual neurons, "candidate" GABAA receptors responsible for the differential actions of ethanol will be identified with correlational analysis. This will allow further testing of the hypothesis that selected GABAA receptor subtypes account for the variable actions of ethanol on GABA responsiveness in the presence of a neurosteroid. For Specific Aim IV, cDNAs for the "candidate" receptor subunits will be transfected into modified HEK cells to validate that neurosteroids will result in the expected response pattern to ethanol on GABA function observed from neurons. Additionally, neurons and cells with a preponderance of type-l-BZD receptors will be transduced with selected GABAA receptor subunit mutants to explore the potential molecular sites responsible for the neurosteroid influence on ethanol's action. Completion of the Aims will allow critical new information to emerge concerning the molecular basis of neurosteroid action on GABAA receptor function that allows ethanol to enhance GABA function in vitro.